As disclosed in U.S. Pat. No. 5,362,634, fermentation product A83543 is a family of related compounds produced by Saccharopolyspora spinosa. The known members of this family have been referred to as factors or components, and each has been given an identifying letter designation. These compounds are hereinafter referred to as spinosyn A, B, etc. The spinosyn compounds are useful for the control of arachnids, nematodes and insects, in particular, Lepidoptera and Diptera species. The compounds are considered environmentally friendly with an appealing toxicological profile.
The naturally produced spinosyn compounds are macrolides consisting of a 21-carbon tetracyclic lactone to which are attached two deoxysugars, a neutral sugar (rhamnose) and an amino sugar (forosamine) (see Kirst et al. (1991). If the amino sugar is not present, the compounds have been referred to as the pseudoaglycone of A, D, etc., and if the neutral sugar is not present then the compounds have been referred to as the reverse pseudoaglycone of A, D, etc. A more preferred nomenclature is to refer to the pseudoaglycones as spinosyn A 17-Psa, spinosyn D 17-Psa, etc., and to the reverse pseudoaglycones as spinosyn A 9-Psa, spinosyn D 9-Psa, etc.
The naturally produced spinosyn compounds may be produced via fermentation from S. spinosa strains NRRL 18395, 18537, 18538, 18539, 18719, 18720, 18743 and 18823 and derivatives therefrom. These cultures have been deposited and made part of the stock culture collection of the Midwest Area Northern Regional Research Center, Agricultural Research Service, United States Department of Agriculture, 1815 North University Street, Peoria, Ill., 61604.
U.S. Pat. No. 5,362,634 and corresponding European Patent No. 0375316 B1 relate to spinosyns A, B, C, D, E, F, G, H, and J. These compounds are said to be produced by culturing a strain of the novel microorganism Saccharopolyspora spinosa selected from NRRL 18395, NRRL 18537, NRRL 18538, and NRRL 18539.
WO 93/09126 relates to spinosyns L, M, N, Q, R, S, and T. Also discussed therein are two spinosyn J producing strains: NRRL 18719 and NRRL 18720, and a strain that produces spinosyns Q, R, S, and T: NRRL 18823.
WO 94/20518 and U.S. Pat. No. 5,670,486 relates to spinosyns K, O, P, U, V, W, and Y, and derivatives thereof. Also discussed therein is spinosyn K-producing strain NRRL 18743.
A challenge in producing spinosyn compounds arises from the need to identify and validate neutral sites in the S. spinosa genome, wherein a polynucleotide containing a gene expression cassette could be integrated and stably expressed. The introduced gene expression cassette can contain biosynthetic genes that provide a method for producing new derivatives of the spinosyns which may have a different spectrum of insecticidal activity or a gene expression cassette which can increase the titer levels of spinosyns, in addition to other gene expression cassettes which would impart new beneficial characteristics to the existing spinosyn production strains. It would be advantageous to identify and introduce genes, which result in increased production of spinosyn compounds. It would also be advantageous to utilize neutral sites, wherein stable integration results in little to no negative impact on spinosyn production, growth or other desired metabolic characteristics.